piggyBac Insertional Mutagenesis Screen Identifies a Role for Nuclear RHOA in Human ES Cell Differentiation

Gayle, Sophia; Pan, Yukun; Landrette, Sean F.; Xu, Tian

doi:10.1016/j.stemcr.2015.03.001

Cited by 12 publications

(9 citation statements)

References 42 publications

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“…To stably overexpress FOXH1 in hESCs, FOXH1 cDNA was amplified by RT-PCR from H1 ESC RNA using the Superscript III First Strand Synthesis System (Thermo Fisher) and the oligonucleotides listed in Table S2, digested with MluI, and cloned into a piggyBac transposon-based vector containing an ACTB promoter and a blasticidin S resistance cassette ( Gayle et al, 2015 ; gift of T. Xu). H1 ESCs were cotransfected with either the FOXH1 or empty piggyBac together with the plasmid Act-PBase using the P4 Primary Cell 4D-Nucleofector X Kit (Program CA137; Lonza).…”

Section: Methodsmentioning

confidence: 99%

The RNA exosome nuclease complex regulates human embryonic stem cell differentiation

Belair

Sim

Kim

et al. 2019

Journal of Cell Biology

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A defining feature of embryonic stem cells (ESCs) is the ability to differentiate into all three germ layers. Pluripotency is maintained in part by a unique transcription network that maintains expression of pluripotency-specific transcription factors and represses developmental genes. While the mechanisms that establish this transcription network are well studied, little is known of the posttranscriptional surveillance pathways that degrade differentiation-related RNAs. We report that the surveillance pathway mediated by the RNA exosome nuclease complex represses ESC differentiation. Depletion of the exosome expedites differentiation of human ESCs into all three germ layers. LINE-1 retrotransposons and specific miRNAs, lncRNAs, and mRNAs that encode developmental regulators or affect their expression are all bound by the exosome and increase in level upon exosome depletion. The exosome restrains differentiation in part by degrading transcripts encoding FOXH1, a transcription factor crucial for mesendoderm formation. Our studies establish the exosome as a regulator of human ESC differentiation and reveal the importance of RNA decay in maintaining pluripotency.

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Section: Methodsmentioning

confidence: 99%

The RNA exosome nuclease complex regulates human embryonic stem cell differentiation

Belair

Sim

Kim

et al. 2019

Journal of Cell Biology

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“…Human PIKFYVE, CTSD, and TFEB complementary DNAs were amplified using Kapa HiFi (Kapa Biosystems) and cloned into the piggyBac transposon construct PJ[puro] 20 MluI site using NEBuilder HiFi Assembly Mix (New England Biolabs). Five million cells were electroporated with a 2:1 ratio of PJ[puro] and ACT-PBase with the Neon transfection system (Thermo Fisher Scientific).…”

Section: Cell Line Transfections and Overexpressionmentioning

confidence: 99%

Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma

Gayle¹,

Landrette²,

Beeharry³

et al. 2017

Blood

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170

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We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes, , and were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.

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“…A piggyBac transposon-based insertional mutagenesis method has been developed to efficiently generate genome-wide mutant libraries in mouse haploid ESCs [275]. In hESCs a piggyBac insertional mutagenesis screen was used to identify the role of nuclear RhoA during stem cell differentiation [276]. The Sleeping Beauty transposon has been also used to explore the clonal dynamics of native haematopoiesis in vivo allowing for a specific fate tracking approach based on in situ labelling of HSCs.…”

Section: Dna Transposons As Tools For Genetic Engineering Of Stem Cellsmentioning

confidence: 99%

The impact of transposable element activity on therapeutically relevant human stem cells

et al. 2019

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Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.

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piggyBac Insertional Mutagenesis Screen Identifies a Role for Nuclear RHOA in Human ES Cell Differentiation

Cited by 12 publications

References 42 publications

The RNA exosome nuclease complex regulates human embryonic stem cell differentiation

The RNA exosome nuclease complex regulates human embryonic stem cell differentiation

Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma

The impact of transposable element activity on therapeutically relevant human stem cells

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